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Bioassay

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Bioassay

  1. 1. BIOASSAY Dr. RENJU.S.RAVI
  2. 2. OVERVIEW  BIOASSAY – Definition/Synonyms  PRINCIPLES OF BIOASSAY  INDICATIONS OF BIOASSAY  TYPES OF BIOASSAY  USES OF BIOASSAY  DRAWBACKS  BIOASSAY IN HUMANS
  3. 3. What is ASSAY  Amount or activity of an active principle in unit quantity of preparation  Types: 3 Physico-chemical assay Biological (Bioassay) Immunological assay Photometry Chromatography RIA ELISA
  4. 4. Bioassay Potency or concentration of an active principle in unit quantity of preparation by measuring its biological response on living tissues Introduced by Paul Ehrlich - biostandardization of Diphtheria antitoxin 4
  5. 5. Synonyms 5 Biological assay Bio metrics Biological standadization Bio-standadizatio n
  6. 6. Principles of bioassay To compare the test substance with the International Standard preparation of the same To find out how much test substance is required to produce the same biological effect, as produced by the standard 6
  7. 7. Contd.. Activity assayed should be the activity of interest Standard & test sample - similar pharmacological effects & mode of action Both should be compared for their established pharmacological effect using specified technique Ex: *Ach – contractile response on frog rectus *Histamine – contractile response on guinea pig ileum 7
  8. 8. Contd.. Problem of biological variation must be minimized  Experimental conditions - kept constant  Animals - same species, sex and weight  Number of animals - large enough to minimize error (individual variation)  Isolated preparations - sensitive 8
  9. 9. Indications of bioassay  No chemical method has been developed  Chemical assay is too complex /not sensitive enough to measure (ex: insulin, Ach)  To measure the pharmacological activity of new or chemically undefined substances  For biological standardization of drugs obtained from natural sources as these cannot be obtained in pure form. Eg: Oxytocin,Vasopressin,Insulin,Heparin.. 9
  10. 10. Contd…  To compare the strength of a drug obtained from various sources due to different compositions (Eg:Cardiac glycosides)  Chemicals with similar structure, but different biological activity  Chemical structure of the active principle is unknown  Chemical structure known; cannot be actively purified. Eg: Peptide hormones 10
  11. 11. Characteristics of a good assay method  Sensitivity  Specificity  Repeatability  Reproducibility  Precision  Accuracy  Stability – tissue has to stay “bioassay-fit 11
  12. 12. Bioassay can be performed on 12 • Intact Invivo animals • Isolated tissues • Specific cells • Organisms Invitro
  13. 13. WHOLE ANIMALS  Nor Adrenaline – Spinal Cat  Cardiac Glycosides – Guinea Pig  Insulin – Mice  Estrogens – Ovariectamised Female Rat MICRO ORGANISMS Vit B12 – Euglena gracilis Tetracycline - Bacillus pumilus 13
  14. 14. ISOLATED TISSUE Acetyl Choline – Frog Rectus Abdominus muscle  Histamine – Guinea Pig ileum  Adrenaline – Rat uterus Oxytocin – Rat uterus oestrogen primed DISPERSED CELLS • Plasma LH estimation by stimulation of testosterone synthesis - on isolated Leydig cells 14
  15. 15. Types of Bioassay QUANTAL ASSAY GRADED ASSAY INDIRECT ASSAY DIRECT ASSAY
  16. 16. Quantal assay  Quantal response - the response is in the form of "all or none", i.e. either no response or maximum response  Drugs producing quantal effect can be bioassayed by end point method 17
  17. 17.  The threshold dose producing a predetermined effect is measured  Comparison between the results of standard and the test  E.g: Bioassay of digitalis in cats,Insulin induced hypoglycemic convulsions in rat Threshold dose of the Std X Conc of Std Threshold dose of the Test Conc of Unknown =
  18. 18. Graded assay  Graded response - response is proportional to the dose and response may lie between no response and the maximum response.  Types: • Bracketing /direct matching • Interpolation • Multiple point assays Three point assay Four point assay Six point assay • Cumulative dose response 19
  19. 19. Graded DRC 20 standard Test/unknown
  20. 20. 21 DRC & Log DRC 70% 30% Sigmoid curve Wide range of doses can plot  Rectangular hyperbola • Potency • Efficacy • Slope of curve
  21. 21. 22
  22. 22. Bracketing or Direct Matching  A constant dose of the standard is bracketed by varying dose of test sample  until an exact matching between the response of std & that of the sample is achieved  Strength of unknowm/test drug can be found by simple interpolation of bracketed response. 23
  23. 23. 24
  24. 24. Bracketing or Direct Matching 25
  25. 25. ADVANTAGES  Simple & Faster  Amount of test drug available is small  Does not involve complicated calculations  Does not depend on DRC 26
  26. 26. Disadvantages  less accurate,time consuming, troublesome  cannot get exact match of response  quantitative difference b/w test & std not obtained
  27. 27. Interpolation assay  A log dose-response curve is plotted with the standard on a simple graph paper or Semi-log paper  The concentration of the test is then read from the graph 28
  28. 28. 29 standard Test/unknown
  29. 29. Interpolation 100 % standard R E S 50 P O N S E 0 x x1 LOG DOSE
  30. 30. Advantages  Sensitivity of tissue is 1st determined by prior plotting of a conc-response curve with known agonist  Dose can be plotted even if it varies over thousand fold range  Error is normally distributed
  31. 31. Disadvantages  Sensitivity of tissue changes with time  Timing of doses not taken into account  Variation in mode of application of drugs
  32. 32. Multiple point assays  Responses are repeated several times and the mean of each is taken  Chances of error are minimized  3 point method - 2 doses of std+1 dose of test  4 point method - 2 doses of std+2 doses of test  6 point method - 3 doses of std+3 doses of test  Latin square method of randomization to avoid any bias 33
  33. 33. 34 t s1 s2 s1 s2 t s2 t s1 3 - point assay 3 cycles
  34. 34. 3-POINT ASSAY % R E S P O N S E LOG DOSE 35 s1 T t S2 s2 S1
  35. 35. Latin square design s1 s2 t t s1 s2 s2 s1 t t s2 s1
  36. 36. CALCULATION • Mean responses of these 3 sets plotted • Log potency ratio (M) = (T-S1÷ S2-S1)× log d where, d – dose ratio = s2/s1 • Strength of unknown = s1/t × antilog of M 37
  37. 37. 38 4 - POINT ASSAY
  38. 38. 4-POINT ASSAY % R E S P O N S E LOG DOSE 39 s1 T1 t1 S2 s2 S1 T2 t2
  39. 39. Latin square design s1 s2 t1 t2 s2 t1 t2 s1 t1 t2 s1 s2 t2 s1 s2 t1
  40. 40. Calculation • Mean responses of 4 sets plotted • Log potency ratio (M) (T2-S2)+(T1-S1) × Log d (S2-S1)+(T2-T1) where, d-dose ratio = s2/s1 • Strength of unknown = s1/t1 × antilog of M 41
  41. 41. Six point assay  3+3 dose assay  3 conc each of std & test drug are used  6 sets of experiments using 6 doses in each set  More time consuming,lesser in use  Reliability is excellent
  42. 42. Cumulative Dose Response Curve  Increase conc of drug in bath fluid step by step without washing out the preceeding doses  Continue till supramaximal effect is seen  Dose response curve is plotted
  43. 43. Cumulative dose response
  44. 44. Uses of Bioassay  to measure the pharmacological activity of new/ chemically undefined substances  to investigate the function of endogenous mediators to measure drug toxicity and unwanted effects  to measure the conc of drugs and other active substances in the blood or other body fluids 45
  45. 45.  Determination of potency, ED50/LD50 of drugs  New drug development  Measure clinical effectiveness 46
  46. 46. Drawbacks • Biological variation • Troublesome • Time consuming • Expensive • Less accurate than physico-chemical methods 47
  47. 47. HUMAN TISSUE BIOASSAY • Veins [surgery on varicose veins] • Tissues like larger blood vessels obtained during amputation • Organs removed during transplantation/ tumor surgeries/procedures requiring resection • Tissues collected Postmortem 48
  48. 48. Conclusion  Successful tool in estimation & discovery of biologically active substances  Sensitivity & Specificity – important tool in pharmacology

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